WO2004105734A1 - Procede de preparation de microcapsules - Google Patents

Procede de preparation de microcapsules Download PDF

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Publication number
WO2004105734A1
WO2004105734A1 PCT/CA2004/000803 CA2004000803W WO2004105734A1 WO 2004105734 A1 WO2004105734 A1 WO 2004105734A1 CA 2004000803 W CA2004000803 W CA 2004000803W WO 2004105734 A1 WO2004105734 A1 WO 2004105734A1
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Prior art keywords
microcapsule
particle
particles
microns
coating
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PCT/CA2004/000803
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English (en)
Inventor
Patrice Hildgen
Jean-Michel Rabanel
Philippe Mercier
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Valorisation Recherche, Societe En Commandite
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Publication of WO2004105734A1 publication Critical patent/WO2004105734A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin

Definitions

  • the present invention relates to a method of preparing permeable and sized microcapsules having suitable mass transfer capabilities. More precisely, the present invention relates to a method of preparing permeable and sized microcapsules having suitable mass transfer capabilities, wherein a polymeric degradable particle is first prepared, then coated with a particular coating mixture, and finally selectively degrading the coated particle obtained in the previous step so as to form a microcapsule containing a cavity.
  • the so obtained microcapsule may optionally be loaded with biologically active ingredients and may be used for cell encapsulation or as drug delivery system.
  • Immuno- isolation also allows for the use of animal cells lines, known as xenotransplantation, and immortalized cell lines, eventually genetically modified, instead of primary human cell lines.
  • the active molecule can be continuously secreted or in response to host stimulus, for example glucose concentration for encapsulated pancreatic islets.
  • Other advantages of encapsulation consist in the possibility to target specific organ or body compartments, thus minimizing systemic dosage and potential side effects.
  • the capsule can be considered as a "niche", a cell friendly microenvironment with the presence of an extra-cellular matrix, which can serve as cell scaffold. The use of such a capsule can also prevent excessive cell growth and eventually allow for the removal of the cells if problems arise during the course of treatment.
  • Macroscopic devices (more than 1 ,5 mm) are usually implanted by surgical procedures (idem. Uludag et al. (2000)). Macrocapsules are usually formed with synthetic co-polymers such as: polyacrylonitrile-polyvinyl chloride (PAN-PVC), polyethersuflone (PES), poly-tetra-fluoro-ethylene (PTFE) or polypropylene (PP) for greater stability (Advanced Drug Delivery Reviews, 33, (1 998), pp. 87-1 09, Li, R.H.). These devices are known for their stability, high loading capacity and retrievability, though a problem associated with them consists in that mass transfer is not optimal.
  • PAN-PVC polyacrylonitrile-polyvinyl chloride
  • PES polyethersuflone
  • PTFE poly-tetra-fluoro-ethylene
  • PP polypropylene
  • Fibrous tissue deposition is frequent and the size of the device makes implantation invasive and lowers a patient comfort. Moreover, the constant need for surgical procedures can impose a recurrent risk of infection.
  • micro-encapsulation systems under the 1 mm range have been described in earlier works, such as in the scientific journals entitled: Sciences, 146, (1 964), pp. 524-525 (Chang T.G.) and Science, 210, (1 980), pp. 908- 910 (Lim F., et al.). Most of these micro-encapsulation systems rely mainly either on gentle hydrogel cell embedding or synthetic membrane formation by co-extrusion of a cell preparation and capsule material.
  • Natural polyelectrolyte polymers such as alginates can form gel when in contact with electrolytes of opposite charge such as cations, namely Ca 2+ , Ba 2+ or poly-L-Lysine, and they can form capsules by emulsification or extrusion (see Science, 210, (1 980), pp. 908-91 0 (Lim F., et al.)).
  • synthetic hydrogel polymers such as polyphosphazene, or blends of polymethacrylates have been proposed as well as the combination of natural and synthetic polymers. Use of agarose, forming gel upon heating, has been described for bead preparation.
  • micro-encapsulation mass transfer of various products such as molecules secreted by encapsulated cells, nutriments, drugs or products of enzymatic reactions, is optimized (high surface/internal volume ratio); thus increasing cell viability and allowing a faster secretory response to an external signal.
  • a micro-encapsulation technique also has its limitations. These limitations include mechanical fragility and instability of ionic interactions in hydrogels and batch-to-batch variability, which leads to differences in permeability properties. This is particularly the case for natural hydrogels, wherein such materials entail problems of variable biocompatibility of materials, limited cell loading capacity, stress on cells during encapsulation procedures and non homogenous repartition of cell in beads which could eventually lead to host immunological responses.
  • Other limitations are further described in an scientific article entitled “Engineering challenges in cell- encapsulation technology", Trends in Biotechnologies, 14, (1996), pp. 158- 161 (Colton C.K.).
  • International publication no. WO 02/05943 A1 distinguishes itself from the present application in that it describes a process for preparing a gas during amylopectin reticulation by bubbling.
  • the so described process is very different from the one described in the present invention, namely in that it does not suggest or disclose the possibility that active ingredients can be incorporated in the polymeric degradable particle.
  • the process defined in the aforementioned international publication does not work. Indeed, the only feature that seems to work in this International publication is automatic cell injection. Few methods have been described to prepare hollow particles in the micrometer and millimeter range and which are compatible with cell viability.
  • a first object of the invention is to satisfy the above needs.
  • the invention provides a process for preparing a permeable and sized microcapsule having suitable mass transfer capabilities, said method comprising the step of: a) preparing a polymeric degradable particle and optionally loading it with an active ingredient. b) coating said polymeric degradable particle obtained in step a) with a coating mixture by emulsion polymerization, thus forming a coated particle of desired thickness; and c) selectively degrading the coated particle obtained in step b), in either basic or acidic conditions, by a pH change and/or hydrolytic degradation so as to form at least one sized cavity inside said coated particle.
  • An advantage with the above-mentioned method for preparing microcapsules resides in that the size of the cavity can be varied. Indeed, other components such is size, morphology and porosity of the particle may also be changed.
  • a second object of the invention lies in a permeable and sized microcapsule having suitable mass transfer capabilities as obtained by the method mentioned hereinabove.
  • a third object of the invention lies in the use of a permeable and sized microcapsule having suitable mass transfer capabilities as prepared by the method as defined hereinabove for cell encapsulation, for the preparation of microreactors and/or microreservoirs, and the preparation of multi- compartmental capsules.
  • FIG. 1 is a schematic illustration of the products obtained during the steps of the method for preparing microcapsules according to the present invention.
  • step (1 ) represents a polymeric degradable particle that has been prepared in this step
  • step (2) represents the polymeric degradable particle that is obtained after coating in step 2
  • step (3) represents the so coated polymeric degradable particle after it has been subjected to selective degradation to form a cavity therein
  • step (4) represents the microcapsule loaded with cells and/or active ingredients.
  • FIGS. 1 are illustrations of microcapsules obtained by the method according to the present invention: A) Core particles containing amylopectin particles crosslinked with 10% (w/w) trimetaphosphate (TMP), stained with chromium oxide powder, size: 250-355 micron (in 0.05 M NaOH); B) idem particle obtained after coating with an amylopectin gel crosslinked with epichlorohydrin (particles sieved: 425-500 microns); C) core particle subjected to selective degradation, assay: incubation, NaOH 1 N/37°C/48 hours, core particles are stained with methylene blue. Bar represents 0.500 mm.
  • Figure 3 are illustrations of the PLGA core particles after they have been subjected to the steps of coating and selective degradation according to a preferred embodiment of the invention (Assay PGE0003).
  • Figure 4 are illustrations of particles when they have been subjected to the step of coating according to the present invention and the effect of Span 80TM therein.
  • Figure 5 are illustrations of particles when subjected to the coating according to a preferred embodiment of the present invention and the effect of polyethylene glycol distearate (PEGDS) and viscosity thereon.
  • Figure 6 are illustrations of core particles after having been subjected to the step of coating according to a preferred embodiment of the present invention.
  • Figure 7 are illustrations of microcapsules obtained by the method according to the present invention. The illustrations are by a SEM.
  • Control amylo-pectin gel particle Assay C001 :
  • Figure 8 are illustrations of microcapsules obtained by the present invention and depicting the release of fluorescent markers.
  • Figure 9 is a graph demonstrating the diffusion of fluorescent markers.
  • Figure 1 1 are illustrations of microcapsules obtained by the method according to the present invention, which are to preferably be used as a microreservoir ( Figure 1 1 A) or a microreactor ( Figure 1 1 B).
  • core particle and “template particle” may be used to define the “polymeric degradable particle” as used in the appended claims.
  • mass transfer as defined by the IUPAC Compendium of chemical terminology, relates to a “spontaneous process of transfer of mass across non-homogeneous fields.
  • the driving force can be difference in concentration (i.e. diffusion gradient) or partial pressure of the component.”
  • the expression “mass transfer” may also relate to the diffusion of substances that could either penetrate inside the microcapsule, for example gas, nutriments, reactive agents, etc., or get out of the microcapsule, for example via secretion.
  • the object of the present invention is to provide a method of preparing a permeable and sized microcapsule having suitable mass transfer capabilities.
  • This method comprises the steps of: a) preparing a polymeric degradable particle and optionally loading it with an active ingredient, b) coating said polymeric degradable particle obtained in step a) with a coating mixture by emulsion polymerization, thus forming a coated particle of desired thickness; and c) selectively degrading the coated particle obtained in step b), in either basic or acidic conditions, by a pH change and/or hydrolytic degradation so as to form at least one sized cavity inside said coated particle.
  • a polymeric degradable particle is prepared.
  • a polymeric degradable particle can be prepared by various techniques known to a person skilled in the art.
  • polymers that can be used as choice of polymeric degradable particle according to the present invention include, but is not limited to: polyesters, polyanhydrides, polyamides, polyorthoesters, polyacrylcyanides, polylactide, poly(lactide-co- glycolide), polycaprolactone, polyhydroxybutyrate, their copolymers and mixtures thereof.
  • polyesters polyanhydrides, polyamides, polyorthoesters, polyacrylcyanides, polylactide, poly(lactide-co- glycolide), polycaprolactone, polyhydroxybutyrate, their copolymers and mixtures thereof.
  • suitable polymers and combinations thereof which can be used as choice of polymeric degradable polymer particle.
  • the polymeric degradable particle such as PLA and/or PLGA
  • the Applicant can prepare a coated particle of desired thickness.
  • this step comprises coating of the polymeric degradable particle obtained in the preceding section.
  • a natural polysaccharide such as an amylopectin is dissolved in a NaOH solution under stirring so as to form a solution.
  • epichlorohydrin and/or another crosslinking agent known to a person skilled in the art is then added to this solution, thus forming a coating mixture.
  • the polymeric degradable particles being treated for example by sieving, are then added to the aforementioned coating mixture and is preferably incubated for a few minutes.
  • the viscous mixture is then preferably introduced into paraffin oil under stirring.
  • the solution can further contain a surfactant, though such is not necessarily required.
  • paraffin oil can by replaced by another type of oil such as silicone oil.
  • paraffin oil is preferred since it is non-toxic and inert.
  • introduction of the viscous mixture into the paraffin oil can be done by a direct transfer or by injection.
  • suitable types of polysaccharides may include and is not limited to starches, modified starches, alginate, amylopectin, cellulose, amylose, chitosan, xanthan and other modified celluloses. There, of course, exist other types of natural polysaccharides that may be considered.
  • the crosslinking agent used in step b) may include trimetaphosphate, epichlorohydrin and other chemical compounds that are accepted by the FDA standards.
  • reticulating agents such as: dichlorodiethyl ether; dibasic/tribasic carboxylic acid (both carboxyl etherify OH groups); anhydrides (acetic); divinyl sulfone; diepoxides; cyanuric chloride; di-isocyanates; 1 ,6-hexanedibromide; N,N methylenebisacrylamide; esters of propynoic acid; imidazolium salts of polybasic carboxylic acids; aldehydes such as formaldehyde, acetaldehyde, dialdehyde, glutaraldehyde (toxic); and N,N-(3-dimethylaminopropyl)-N-ethyl carbodiimide (EDC), may be used.
  • the coated particle obtained in step b) preferably has a thickness of about 75 to 100 microns.
  • the thickness could preferably be from 500 nm to 500 microns.
  • the coated particle obtained in section B) is selectively degraded, in either basic or acidic conditions, by a pH change and/or hydrolytic degradation so as to form a sized cavity inside the coated particle.
  • reaction wherein one changes pH of a medium and/or hydrolytic degradation is quite well known to a person skilled in the Art.
  • it is beneficial to use such a step in that it allows the Applicant to selectively adjust the size of the cavity in the coated particle, in either basic or acidic conditions, thus allowing the microcapsule to contain foreign matter.
  • microcapsule it is at this stage in the method of preparing a permeable and sized microcapsule having suitable mass transfer capabilities, is that one can modify the nature of the microcapsule. For example, one can:
  • microcapsules obtained by the method according to the present invention will depend on the applications considered.
  • a microcapsule with one or more compartments can be used for the insertion of cells into an artificial organ application or in a biotechnological application.
  • Microcapsules can also be used as a microreservoir system, which is able to liberate drugs at a constant drug rate.
  • the drug can be a protein, genetic material, a peptide and/or small molecules.
  • suitable drugs may include and are not limited to: plasmids for gene therapy, proteins (i.e. deficient enzymes, G-CSF, erythropectin, growth hormones, FSH and the like), and those known to person skilled in the art to have low biodisponibility or stability problems.
  • proteins i.e. deficient enzymes, G-CSF, erythropectin, growth hormones, FSH and the like
  • drugs may include: omeprazole, enanapril and the like.
  • microcapsules obtained by the method according to the present invention can also be used as a drug delivery system. It is worth mentioning that in the application of oral drug delivery, the goal of the microcapsule is to increase the biodisponibility of the drug. This goal can be achieved since the coating of the microcapsule acts as a mucoadhesive agent.
  • the surface of the microcapsule becomes porous.
  • the microcapsules containing one or more cavities; thus being multi-compartmental may also contain a reactive material. Indeed, when the microcapsule erodes, the reactive material can diffuse into a cavity where a reaction can occur and form an active material, such as NO. It is worth mentioning that the active material can also diffuse outside of the microcapsule. In fact, the control of drug release is obtained by the porosity and pore size of the microcapsule. Preferably, the release rate is a near zero order mechanism.
  • the mean size of core particle obtained from the above- mentioned techniques is preferably from 500 nanometers to 500 micrometers.
  • microcapsule obtained by the method according to the present invention can also be used as a micro- reactor.
  • Polylactide was prepared in the laboratory. Briefly, a catalyst such as tetraphenyl tin (Aldrich) was mixed with a precursor 3,6-dimethyl-1 ,4- dioxane-2,5-dione (Aldrich) in a 1 /10 000 proportion (w/w) and introduced in a round bottom flask under a continuous flush of an inert gas such as argon. Preferably, the flask was heated at 180°C for 3 hours. By-products were dissolved in acetone and precipitated in water. The polymer was dried and freeze-dried.
  • a catalyst such as tetraphenyl tin (Aldrich) was mixed with a precursor 3,6-dimethyl-1 ,4- dioxane-2,5-dione (Aldrich) in a 1 /10 000 proportion (w/w) and introduced in a round bottom flask under a continuous flush of an inert gas such as argon. Pre
  • Polylactide-co-glycolide (PLGA) was from Boehringer- Ingelheim (RG 504H, 50:50 lactide/glycolide) with a Mw of 48 000, partially hydrolysed amylopectin (Glucidex 2) was from Roquette (France); paraffin oil, Epichlorohydrin and Span 80TM were from Fluka; fluorescein isothiocynate (FITC) dextrans, trimetaphosphate (TMP), sodium carbonate and polyethylene glycol distearate (PEGDS) were from Sigma, organic solvents were from Laboratoire MAT (Montreal), partially hydrolyzed polyvinyl alcohol (9-10000 PM) was from Aldrich.
  • SEC Size Exclusion Chromatography
  • trimetaphosphate and 20 g of amylopectin were stirred in 36 ml of H 2 O for 2 hours. 4 ml of sodium carbonate was added and the preparation was incubated for 90 minutes at 42°C with frequent agitation. The preparation was transferred slowly into 600 ml of paraffin oil under stirring (750 rpm, Caframo mixer) overnight at 25°C. Water was added and after sedimentation, the particles were washed, re-dispersed in 0,05 M NaCI and sieved. Coating of amylo-pectin core particles
  • amylopectin was dissolved in 6 ml of NaOH (2N).
  • epichlorohydrin ratio: 1 mole per 1 ,5 mole of sugar monomer
  • 10g of core particles are mixed and transferred into 600 ml of paraffin oil under stirring (500 rpm, Caframo mixer) for 6 hours. Particles were then recovered by sedimentation in water, sieved (mesh sizes: 150, 355, 425 and 710 microns) and washed to remove salts, traces of oil and epichlorohydrin.
  • PLA and PLGA core particles were prepared according to the solvent extraction-evaporation technique. Typically, 500mg of PLGA or PLA were dissolved in 3,5 ml of chloroform and injected in 250 ml of 0,3% PVA (w/v) and kept under stirring for 12 hours (Caframo stirrer). Particles were then collected, sieved (sieves: 150 and 350 microns) and washed before coating. Viscosity of the polyester solution in solvent (5-20% w/v) and stirring speed (150 to 350 rpm) were adjusted for desired particle size.
  • amylopectin Glucidex 2
  • 2,5 to 7 ml of NaOH (2N) for one hour under stirring.
  • epichlorohydrin was added in a ratio of 1 /1 ,5 or 1 /9 (ratio mole of epichlorohydrin per mole of sugar monomer).
  • Sieved core polyester particles were added and incubated under stirring for 1 to 5 minutes.
  • the viscous mixture was then introduced in 400 to 800 ml of paraffin oil under stirring (straight blade or Rushton propeller, at a speed of 250 to 900rpm, Caframo stirrer) in the presence of surfactant Span 80TM (from 0% to 0,5% vol.
  • the surfactant is only a preferred embodiment of the invention. Indeed, the preferred introduction of the surfactant can be either done by direct transfer or by injection. In the latter case, the mixture was introduced in a 5 ml syringe and injected by a syringe pusher (Harvard 1 1 ) at a velocity of 45 ml /hr. The needle (18 Gauge) was completely immerged in paraffin oil. Particles were recovered by sedimentation in water, sieved (mesh sizes: 150, 355, 425 and 710 microns) and washed to remove salts, traces of oil and epichlorohydrin. Drained particles fractions were weighed to calculated yield.
  • the coated PLA or PLGA particles can be suspended in NaOH (1 N) solution and incubated at 37°C for 24 to 96 hours. Particles are collected, sieved and washed in MilliporeTM water before further use (fraction 425 to 710 microns retained for analysis).
  • Microphotographs were recorded with digital camera using Northern Eclipse software and analyzed for particle size with Optimas (v6.0) image analysis software and examined for morphology by visual inspection. Scanning electron microscopy
  • Control particles and final hollow particles fractions were examined on a JeolTM scanning electron microscope.
  • Fluorescein isothiocyanate Dextrans (FITC-Dextrans) were introduced in polyester core particles as an emulsion. 50 ⁇ l of a FITC-Dextrans aqueous solution was added to the polyester/chloroform mixture, sonicated for 20 seconds on a 550 Sonic DismenbranorTM (Fisher). This primary emulsion was then transferred in a 250 ml 0,3% PVA under stirring (see section annotated Preparation of PLA and PLGA core particle). The ratio of polyester/FITC- Dextrans was about 1 mg to 500 mg, except for batch PLE034: 10 mg to 500mg.
  • microparticles were incubated in NaOH (1 N) solution at 37°C, and examined under microscope, on a Zeiss Axiovert S100TM microscope (Zeiss, Germany), which was equipped with a set of emission/excitation filters (excitation: 495nm, emission 517nm) and a fluorescence lamp. Digital pictures were recorded with a digital camera and use of Northern Eclipse software was also made.
  • the 425 to 710 micron fraction of coated particle batch PLE020 was incubated in NaOH (1 N) solution for 72 hours at 37°C to selectively degrade the core particles. After sieving and repeatedly washing, 500mg of the fraction was re-suspended in 1 ml of H 2 0 containing the fluorescent probe (FITC-Dextrans, 4kD, 10kD or 70 kD) and incubated at room temperature. Aliquots of 10 ⁇ l were collected and analyzed on a spectro- fluorometer. A control assay containing 1 ,5ml of H 2 O with the same quantity of fluorescent probe was also incubated in the same conditions.
  • FITC-Dextrans fluorescent probe
  • Fluorescence diffusion results were expressed as a percentage of the control at each time point in time for each of the FITC Dextrans tested. Similar experiments were simultaneously conducted and particles were observed under fluorescence on a Zeiss Axiovert S100 microscope (Zeiss, Germany) equipped with a set of emission/excitation filters and a fluorescence lamp. Pictures were recorded with a digital camera and use was made to Northern Eclipse software.
  • Amylopectin core particle preparation and coating Assays with amylopectin core particles Amylopectin core particle preparation and coating.
  • an amylopectin particle crosslinked with a crosslinking agent such as trimetaphosphate (TMP), also referred to as AP/TMP particle were prepared to be used as "template” or "core” particle, according to the preparation scheme represented in Figure 1 .
  • TMP crosslinked amylopectin (AP) matrix is very sensitive to basic conditions. To illustrate this point a AP/TMP particle of about 500 microns is completely degraded in NaOH (1 N) in less than 2 hours at room temperature.
  • an epichlorohydrin crosslinked amylopectin matrix is much more resistant to basic conditions. Coating of the core AP/TMP particle by emulsion polymerization with an amylopectin/epichlorohydrin gel gave good results. This result is shown in Figure 2B.
  • the size of the core particle must correspond to the size of the cavity expected, for example around 300 microns for assays of amylopectin/TMP core particles (see Figure 2).
  • PLA or PLGA particles were prepared according to the solvent emulsion/evaporation technique described in the Journal of Controlled Release, 17, (1 991 ), pp. 1 to 22 (Arshady, R.), to produce large particles of about 350 microns.
  • the coating of polyester core particles was first carried out by using the same protocol developed to coat amylopectin/TMP core particles.
  • the thickness of the coating was then determined to be about 500 nm to 500 microns to compromise between mechanical stability and mass transfer of various compounds such as nutriments, reactive substances, secreted substances, enzymes, drugs, genetic material, etc.
  • Coating was carried out by an emulsion polymerization of a solution of amylopectin and polyester core particles in paraffin oil under stirring.
  • the coating was stabilized by an epichlorohydrin crosslinking reaction described in Starch, 37, (1985), pp. 297-306 (Kartha et al.). Although some coated polyester particles were obtained in the initial experiments, the yield is very low. The results can be seen in Figure 3. As a result, there was formation of big lumps composed of polyester particles embedded in an amylopectin matrix.
  • the cavities thus obtained were larger than the core polyester particles. This phenomenon can be explained by a partial degradation of the polyester template particle when it is in contact with the strongly basic amylopectin gel and/or mechanical action of the particle under stirring and/or swelling of the capsule under washing at the end of the process. Similar results are also obtained with PLA core particles (data not shown).
  • Polyester particles are degradable in both acidic and basic conditions. Preliminary experiments on uncoated polyester particles confirm that basic conditions are the fastest. This has been confirmed by a scientific article published in the Journal of Microencapsulation, 3, (1986), pp. 203-212 (Makino et al.). It has been further reported that reactions conducted in basic conditions at 37°C are at least two times faster than those conducted at room temperature. Degradation of PLA particles are 4 to 5 times slower than PLGA (Resomer RG 504HTM) particles (data not shown). The coating of the core particle does not affect the extent of the degradation. The end result, as shown in Figure 3, shows hollow particles with residual polyesters after 6 hours for PLGA and 48 hours for PLA.
  • the preliminary experiments also confirm the feasibility in preparing hollow particles by this method and point out the parameters to be optimized, such parameters including core particle size, selective polyester hydrolysis and core particle coating reaction.
  • the Applicant was able to identify the main factors affecting the efficiency of the polyester core particle coating. These factors may include: stability of emulsion in paraffin oil, stirring speed, geometry of mixer, viscosity of amylopectin mixture, interaction between the polyester's surface and the amylopectin mixture. It is worth mentioning that PLA, as choice of polymer for the core particle preparation was done in an attempt to reduce the cost of future preparations.
  • polyethylene glycol distearate can preferably used as a surface modifier.
  • PEGDS is composed of two stearate moieties at each of its extremities and contains a spacer of PEG (Mn around 930).
  • the surface modifier could also have different properties according to its density. It has further been documented in the International Journal of Pharmaceutics, 174, (1 998), pp. 101 -109 (Lacasse et al.) that a 0,1 % concentration of PEGDS inhibits polyester particle adhesion to macrophage but not 1 %.
  • the initial viscosity is the main factor to influence the coating yield, not the surface modifier as seen in Figure 5C.
  • the viscosity of the mixture is the function of not only the water content but also of the extent of the reaction of epichlorohydrin, primarily governed by the incubation duration and the incubation temperature. These parameters are likely to influence not only the coating yield but also the permeability properties the gel capsule walls.
  • the core particle must have the same diameter as the cavity to be formed, i.e. preferably about 1 to 500 microns.
  • the core particle must have the same diameter as the cavity to be formed, i.e. preferably about 1 to 500 microns.
  • particle sizes of about 175 to 200 microns is the ideal size for cell encapsulation applications only, as cavity diameters are around twice the core particle diameter, as seen in Figures 3 and 4.
  • Core particle synthesis was adapted accordingly.
  • the main parameters to be adjusted were the viscosity of the starting polyester/organic solvent solution and the stirring speed of the emulsion.
  • Sizes of the particles were assessed with Optimas ® image analysis software, Area Equivalent Diameters and Ferret diameters measured gave typical results of 190 ⁇ m with a standard deviation (SD) of + /- 35 ⁇ m after optimization of the parameters such as viscosity, stirring speed and PVA %.
  • SD standard deviation
  • polyester particles are not dried; instead they are just washed and sieved before use. Dried particles are rapidly degraded when , in direct contact with strongly basic amylopectin/epichlorohydrin reaction mix. Observation of PLA particles in the amylopectin mixture before crosslinking occurs, show a thin layer of water around each PLA particle (see Figure 6B), unlike the amylopectin gel particle in the same situation (see Figure 6A). It is further shown that the surface of polyester particles has at least some affinity for water. This phenomenon can also be attributed to residual PVA molecules adsorbed on the particle surface. The water layer acts as a shield between the polyester and the basic solution (NaOH 2N).
  • polyester particles containing FITC-Dextrans 70kD (batch PL034) and as can be seen when comparing Figures 6B and 6C, fluorescence can be observed in the water layer surrounding the particles. This could be explained by the segregation of hydrophilic molecules at the surface of the particle as already observed with PEG (see the International Journal of Pharmaceutics, 174, (1998), pp. 101 -109 (Lacasse et al. 1998)). Also, one can expect some polyester hydrolysis at the surface of the particle; the latter inducing release of some fluorescence in the water layer. The presence of this water layer and possible surface erosion of core particle could contribute to the size of cavities observed relative to core polyester particle diameter, as seen in Figures 3 and 5. Optimization of selective degradation
  • the so formed polyester core particles are very compact, and such can be attributed to the high viscosity of the initial polyester/chloroform mix.
  • the choice to use PLA instead of PLGA increases the incubation time necessary to completely degrade the core particle. The latter can take up to 96 hours for PLA.
  • optimization will need to be carried out. Methods such as double emulsion to weaken polyester particle structure and a choice of a PLA with lower molecular weight should be considered, as per the Journal of Controlled Release, 30, (1994), pp. 161 -173 (Park, T.G.).
  • the hydrated particles were examined by scanning electron microscopy at room temperature and under vacuum. Unlike frozen samples, the amylopectin gel undergoes dehydration over time and eventually collapses in these conditions.
  • Control particles made of crosslinked amylopectin (non hollow particles), were first examined (see Figure 7). As seen in Figure 7A', the pictures taken in the initial moments show a smooth surface for native particles. Fractions with hollow particles were then analyzed, before and after polyester core particle hydrolysis. Upon inspection of Figure 7B', one can observe particles at the initial moment that appear like a deflated balloon.
  • the concentration in the external medium remained at a plateau signifying little or no PLEO23 and/or PLE020 diffusion into the particles as expected.
  • 4kD and 10kD markers showed decrease in fluorescence, but slower than expected.
  • the value of fluorescence is greater than the expected value, and such varies between batches.
  • One explanation could be attributed to insufficient washing of particles after incubation in basic conditions. Indeed, it has been shown that the release of NaOH in the medium can affect the intensity of fluorescence (see Methods in Enzymology, 174, (1989), pp. 131 -145 (Ohkuma, S.)). This could also account for the initial burst of fluorescence seen in the first 30 minutes.
  • Permeability porosity & diffusion. Permeability and/or porosity of the capsule to proteins are essential characteristics of the microcapsules. One can expect that the control of the porosity will be an important concern for regulatory agencies to allow clinical trials.
  • capsule gel permeation The principles governing capsule gel permeation are similar to those of size-exclusion chromatography.
  • the ability of a protein to cross the capsule wall depends on gel pore size, size and shape of the protein and interactions between the protein and the gel network (ionic or hydrophobic).
  • Molecular weight cut-off determination using FITC-Dextrans has to be cautiously interpreted as gel permeability is not directly dependent on molecular weight (Mw), but rather on physical size and shape of the molecule.
  • Mw molecular weight
  • R ⁇ viscosity radius
  • the novel method for preparing microcapsules thus produces particles with a range of permeability characteristics in accordance with immuno- isolation parameters.
  • the microcapsules obtained by the method according to the present invention can be used as a micro-reactor.
  • several substances can be loaded in different core particles.
  • one batch is prepared with PLA microspheres containing FeSO4.
  • a second batch can be prepared with PLA to yield PLA microspheres containing sodium nitrite.
  • a microcapsule could include particles from the first and second batch. In this case there is no need of degrading the polyester core.
  • a slow diffusion of both reactive in the medium allows the reaction and generates nitrogen monoxide (NO) .
  • the microcapsule thus acts as a micro-reactor thereby slowly generating NO by a constant rate through the hydrogel.
  • the microcapsules obtained by the method according to the present invention are to be used as a multi-compartmental drug carrier, several substances can be loaded into the core particles.
  • one batch can be prepared with PLA microspheres containing anti-inflammatory drugs, while a second batch is prepared with PLA to yield PLA microspheres containing an opioid drug.
  • the microcapsules could include particles obtained from both the first and second batches.
  • the cores could be degraded or not.
  • the microcapsules obtained by the method according to the present invention can also be used as a micro-reservoir drug delivery system.
  • one substance can be loaded into the core microsphere, and the particle can then be coated with the hydrogel.
  • the core is then degraded.
  • the drug is free inside the cavity and can be released by diffusion through the gel at a constant rate.
  • An example of such an application can be for example the use of an anticancer drug for brain delivery.
  • the novel method of preparing permeable and sized microcapsules as set forth in the present application is successful. Morphology, size and porosity of the particles can be adjusted. This novel method can be extended to other materials for core particle preparation and coating. Indeed, the use of a wide variety of polymeric degradable core materials can be used; and thus allow for selective degradation in acidic conditions, for example by using polyorthoester polymers.
  • the coating can then preferably be done with trimetaphosphate (TMP) as choice of crosslinking agent, which produce links that are sensitive to basic conditions, but less sensitive to acidic conditions.
  • TMP trimetaphosphate
  • the advantage of TMP as choice of cross-linking agent is considered to be more acceptable in terms of toxicity.
  • Coating can also be done with other natural or synthetic hydrogel and crosslinking methods.
  • the method according to the present invention could also be useful to create multi- compartmental drug delivery devices, wherein a drug can be trapped in the polyester hydrophobic medium, then released during degradation at physiologic pH while the hydrophilic coating can be involved in hydrophilic drug storage, release regulation and biocompatibility of the devices.

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Abstract

L'invention porte sur un procédé de préparation de microcapsules perméables et calibrées à capacité de transfert de masse appropriée. Ledit procédé comporte les étapes suivantes: a) préparation de particules de polymère dégradable facultativement chargées par un ingrédient actif; b) enduction par émulsion polymérisation desdites particules au moyen d'un mélange enrobant de l'épaisseur désirée; et c) dégradation sélective de la particule enduite dans des conditions acides ou basiques par variation du pH et/ou dégradation hydrolytique, pour y former au moins une cavité. L'invention porte également sur des microcapsules perméables et calibrées à capacité de transfert de masse appropriée obtenues par ledit procédé, et sur l'utilisation desdites microcapsules.
PCT/CA2004/000803 2003-05-28 2004-05-28 Procede de preparation de microcapsules WO2004105734A1 (fr)

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